205

Journal of Physiology (1992), 451, pp. 205-227 With 9 figures Printed in Great Britain

ELEVATED MUSCLE GLYCOGEN AND ANAEROBIC ENERGY PRODUCTION DURING EXHAUSTIVE EXERCISE IN MAN

BY J. BANGSBO, T. E. GRAHAM*, B. KIENS AND B. SALTIN From the August Krogh Institute, University of Copenhagen, Copenhagen, Denmark (Received 30 July 1991) SUMMARY

1. The effect of elevated muscle glycogen on anaerobic energy production, and glycogenolytic and glycolytic rates was examined in man by using the one-legged knee extension model, which enables evaluation of metabolism in a well-defined muscle group. 2. Six subjects performed very intense exercise to exhaustion (EXI) with one leg with normal glycogen (control) and one with a very high concentration (HG). With each leg, the exhaustive exercise was repeated after 1 h of recovery (EX2). Prior to and immediately after each exercise bout, a muscle biopsy was taken from m. vastus lateralis of the active leg for determination of glycogen, lactate, creatine phosphate (CP) and nucleotide concentrations. Measurements of leg blood flow and femoral arterial-venous differences for oxygen content, lactate, glucose, free fatty acids and potassium were performed before and regularly during the exhaustive exercises. 3. Muscle glycogen concentration prior to EXI was 87-0 and 176-8 mmnol (kg wet wt)-f for the control and HG leg, respectively, and the decreases during exercise were 26-3 (control) and 25-6 (HG) mmol (kg wet wt)-1. The net glycogen utilization rate was not related to pre-exercise muscle glycogen concentration. Muscle lactate concentration at the end of EXI was 18-8 (control) and 16-1 (HG) mmol (kg wet wt)-1, and the net lactate production (including lactate release) was 26-5 (control) and 23-6 (HG) mmol (kg wet wt)-'. Rate of lactate production was unrelated to initial muscle glycogen level. Time to exhaustion for EX1 was the same for the control leg (2-82 min) and HG leg (2-92 min). 4. Muscle glycogen concentration before EX2 was 14 mmol (kg wet wt)-1 lower than prior to EXI. During EX2 the muscle glycogen decline of 19-6 mmol (kg wet wt)-' for the control leg was less than for the HG leg (26-2 mmol (kg wet wt)-1). The muscle lactate concentrations at the end of EX2 were about 7-8 mmol (kg wet wt)-1 lower compared to EXI, and the net lactate production was reduced by 40 %. The exercise time during EX2 was 0 35 min shorter for the control leg, while no difference was observed for the HG leg. 5. Total reduction in ATP and CP was similar during the four exercise bouts, while a higher accumulation of inosine monophosphate (IMP) occurred during EX2 for the *

MS 9611

Present address: Human Biology, University of Guelph, Guelph, Ontario, Canada.

J. BANGSBO, T. E. GRAHAM, B. KIENS AND B. SALTIN 206 control leg (0-72 mmol (kg wet wt)-1) compared to the HG leg (0-20 mmol (kg wet wt)-1). There was no difference in leg oxygen uptake or leg oxygen deficit between the exercise bouts. 6. It is concluded that: (a) elevated muscle glycogen does not influence glycogenolytic or glycolytic rates and fatigue; (b) previous intense exercise reduces lactate production and time to exhaustion in spite of recovery being long enough for lactate (pH) and K+ to return to pre-exercise levels. These findings suggest that muscle and blood lactate concentrations are not the only crucial factors for inhibiting anaerobic carbohydrate usage during intense skeletal muscle contractions and the development of fatigue. INTRODUCTION

A consensus has been reached that dietary manipulation which elevates the carbohydrate stores of the body (muscle and liver) affects metabolism during exercise. Even though this results in an enhanced usage of carbohydrate at a given submaximal exercise intensity and an elevation in muscle and blood lactate, exercise endurance performance is improved. Such dietary manipulation was first studied by Christensen & Hansen (1939) who demonstrated that a carbohydrate-enriched diet resulted in a higher respiratory quotient (RQ) and better exercise performance in prolonged exercise when compared to exercise after days with a mixed or fat and protein diet. Some 30 years later, Bergstrom, Hermansen, Hultman & Saltin (1967) used a similar protocol with the addition of muscle glycogen determinations. Metabolism was affected in relation to the magnitude of glycogen storage in the exercising muscles. Exhaustion was postponed in proportion to availability of muscle glycogen and at exhaustion the active muscles were completely glycogen depleted. In short-lasting, heavy exercise, where the rate of 'anaerobic glycolysis' peaks and its relative contribution to the energy yield is paramount, an even closer relationship between availability of glycogen in the active muscle and its utilization could be anticipated. In studies using rats, increased muscle glycogen storage was shown to induce higher glycogenolytic and glycolytic rates (Richter & Galbo, 1986). In fast twitch fibres, glycogen utilization and lactate release were linearly related to the initial muscle glycogen concentration over a 15 min exercise period. However, Spriet, Berardinucci, Marsh, Campbell & Graham (1990) were not able to confirm this, as they found no relationship between muscle glycogen storage and anaerobic glycogenolysis when the intense exercise period was shortened to 1 min. In several studies in man, it has been concluded that the rate of anaerobic glycogenolysis is a function of muscle glycogen content, when above-normal carbohydrate storage is induced (Asmussen, Klausen, Nielsen, Egelund, Technow & T0nder, 1974; Klausen & Sj0gaard, 1980; Maughan & Poole, 1981; Greenhaff, Gleeson & Maughan, 1987, 1988). In contrast, several other investigators have failed to verify these findings (Jacobs, 1981; Symons & Jacobs, 1989; Ren, Broberg, Sahlin & Hultman, 1990; Spencer & Katz, 1991), but agree with the findings of an early study by Saltin & Hermansen (1967). An effect on the rate of anaerobic glycogenolysis was observed in this latter study when the muscle glycogen content was below 30 mmol (kg wet wt)-1, which is less

MUSCLE GLYCOGEN AND AN.\TAEROBIC ENERGY YIELD

207

than half of the normal muscle glycogen stores in man after a mixed diet (see also Hultman & Sj0holm, 1983). There are no obvious explanations for the differences in results either in man or in other species. The design and protocol of the various studies, however, do vary markedly. Further. in many of the studies on man, only blood substrates and metabolites were analysed to evaluate the metabolic response. This limits direct comparisons and may explain why no firm conclusions so far have been reached. Thus, the aim of the present study was to evaluate the effect of above-normal muscle glycogen content on anaerobic and aerobic metabolism during short-lasting, very intense exercise. To achieve variation in muscle glycogen content, dietary manipulation can be used as well as exercise prior to the test exercise. In the present study both approaches were applied. In addition, the exercise model chosen was onelegged knee extensor kicking, which allows for precise quantitative metabolic evaluation as well as relating these metabolic events directly to the work performed and to the level of exhaustion (Saltin, Kiens & Savard, 1986; Bangsbo, Gollnick, Graham, Juel, Kiens, Mizuno & Saltin, 1990). METHODS

Subjects Six, healthy, male subjects ranging in age from 22 to 27 years, with an average height of 182 (range: 177-190) cm, and an average weight of 76 (69-83) kg, participated in the experiment. Five of the subjects had participated in previous experiments of similar design and measurements. All subjects were habitually physical active, but none trained for competition. The subjects were fully informed of any risks and discomfort associated with these experiments before giving their consent to participate. The study was approved by the local ethical committee.

IProcedures Subjects performed one-legged exercise, in the supine position, on an ergometer that permitted the exercise to be confined to the quadriceps muscles (Andersen, Adams, Sj0gaard, Thorboe & Saltin, 1985). All subjects practiced the exercise with each leg several times on separate days before the final experiment was performed, and endurance (time to exhaustion at the same work rate used in the final experiment) for each of the legs was shown to be equal (mean: 2-85 + 0 52) (± S.E.M) and 2-96+0 49 min). On the experimental day a catheter was placed in the femoral artery with the Seldinger technique, with the tip placed 1-2 cm proximal to the inguinal ligament. Since both legs were to be studied, two catheters were put in each of the two femoral veins. The tip of one of the catheters was positioned approximately 8 cm in the retrograde direction, i.e. 10-12 cm distal to the inguinal ligament. This catheter was used for collecting blood samples and for infusing the ice-cold saline. Another venous catheter was placed in the inguinal region with the tip about 1 cm distal to the ligament. The thermistor for measurement of venous blood temperature was inserted through this catheter and was advanced just proximal to the tip. Protocol In the evening 3 davs prior to the experiment, the subjects exercised with one leg (high-glycogen leg; HG leg) for 2-5-3 h. consisting of 1-25 h of continuous exercise followed by intense, intermittent exercise for the next 1-25-1-75 h at work rates comparable to or higher than the work rate used in the experiment. After this, the subjects followed a carbohydrate-rich diet (76 7 + 2 0 % carbohydrate, 12-0 + 2-0% fat, 10-5 + 0-9% protein; total intake (2 days): 36-9 + 2-3 MJ (185 + 11 MJ day-')). The evening before the experiment, the subject carried out the same exercise protocol with the other leg (control leg). After this, the subject did not ingest any food or fluid except water until finishing the experiment the following day.

J. BANGSBO, T. E. GRAHAM,

208

B. KIENS AND B. SALTIN

On the experimental day, placement of the catheters was followed by 30 min of rest in the supine position. Then, the subject warmed up with one of the legs for 10 min at a work rate of 10 W. The choice of legs was randomized. After at least 10 min of rest, the subject exercised the leg at a mean power output of 67-0 W (range: 54-0-78 4 W; kicking frequency: 60 min-') until exhaustion (EX 1). After a 1 h recovery period (RECI) the exhaustive exercise was repeated at the same power output (EX2). Following a rest period of 20 min (REC2) the subject carried out the same exercise protocol with the other leg (Fig. 1). Exhaustion Biopsy Blood flow

4 4

4

4

Leg 1/1l

4 I

4 4

4

44444

4 4

4

4 4 4,

Im

Leg

0

4

4 4 44 4

Control!/X1E21 high glycogen

4 4

4

4

4

Blood samples 4

4 4

4

0-05) from nil.

Oxygen deficit and anaerobic energy production (Table 4; Figs 4, 6 and 7) The leg oxygen deficit was 848 ml 02 equiv for EXI-C, which was similar to the 907 ml 02 equiv for EXt-HG (Table 4). These values were of the same magnitude as

217

MUSCLE GLYCOGEN AND ANAEROBIC ENERGY YIELD

the oxygen deficit of 745 and 891 ml02equiv for EX2-C and EX2-HG, respectively. The latter two values were different (P < 0&05) from each other due to the longer exercise time of EX2-HG, since the corresponding data adjusted to a common exercise time were not (P > 0105) different (686 + 81 and 715 + 51 ml 02 equiv). TABLE 4. Leg energy demand, VO,, and oxygen deficit (A) and muscle lactate accumulation, release and production (B) during the exhaustive exercise bouts (EX1 and EX2) for the control leg (C) and the high-glycogen leg (HG) EX2 EXt C HG (A) Oxygen deficit 2372 2378 Energy demand +526 +508 (ml 02 equiv) 1472 1508 Leg VO (m102) +482 +430 907 868 Leg oxygen deficit +136 +153 (ml °2equiv) (B) Lactate production 15-6 Lactate accumulation 19-3 +2-3 +3-1 (mmol (kg wet wt)-1) 7-2 8-0 Lactate release +116 +P13 (mmol (kg wet wt)-1) 23-6 Lactate production 26-5

(mmol (kg wet wt)-1)

+2-3

+3-1

C

HG

2003 +375

+405

1258 +354 745 +62

1328 +353 891 +94

11P2

2218

8-5

+ 3-6t 4-6 +0-4t 15-8

± 1Htt

+3-6 t

±Hitt

6-3 _14 14-9

Means +_.E.M. are given. t Significant (P < 0 05) difference between EXt-C and EX2-C. tt Significant (P < 0 05) difference between EXI-HG and EX2-HG.

The changes in nucleotides corresponded to an ATP production of 1'7 ±04 (EX1C) and 1P3+±03 (EX1-HG) mmol (kg wet wt)-t, and 1P9+0-5 (EX2-C) and 1P3±03 (EX2-HG) mmol (kg wet wt)-'. This ATP release together with the net depletion of CP could account for 1119 (EXI-C) and 12-4 (EXt-HG) mmol (kg wet wt)-t, or 20'1 and 19-8°% of the total anaerobic energy production estimated from the oxygen deficit (including use of Mb- and Rb-bound 0w), while the corresponding values for EX2-C and EX2-HG were 11 7 (225 %) and 1416 (23'8 %) mmol (kg wet wt)f', respectively (Fig. 6). The net lactate production was 26-5 and 23-6 mmol (kg wet wt)-1 during EXt-C and EXt-HG, respectively. During EX2-C (15-8 mmol (kg wet wt)fl) and EX2-HG (14-9 mmol (kg wet wt)-') the production was similar, but each was lower (P < 0 05) than the corresponding value for EXt (Table 4; Fig. 4). For EXt-C and EXt-HG, the mean net lactate production rate was 1141+2-0 and 9t1+1t4mmol(kg wet wt)- min-t, respectively, which was higher (P < 0 05) than during EX2-C (7-5+2-3mmol(kg wetwt)-lmin-) and EX2-HG (5-9+019mmol(kg wetwt)-' min-'). Neither the total lactate production nor the mean lactate production rate were related to the pre-exercise muscle glycogen concentration (Fig. 7). In the first 8-2

218

J. BANGSBO, T. E. GRAHAM, B. KIENS AND B. SALTIN

EX1

EX2

70 70 60-

O 50E E

40-

0

colcoe

lyoe

0

20 P-Control

C

Control High

High

glycogen

glycogen

Fig. 6. Total anaerobic energy production during EXI (left) and EX2 (right) determined

.5 deficit 30v related to ATP release from lactate production (0)c and from the leg oxygen changes in OP and nucleotides (EI) (enL others). In the calculation is used: 1 mmol lactate

-C5 mmol ATP 6w7 Ml 02 equiv. 10

o 0

40

.0* 0

X

5

0~~~~

~30

0

5

0

5

v

E E

Lu

c 0

'7

V.

20. 10 IL)

o

-J

0

50 100 150 200 250 Pre-exercise muscle glycogen (mmol (kg wet wt)-1)

Fig. 7. Individual relationship between pre-exercise muscle glycogen concentration and lactate production during EXt (open symbols) and EX2 (filled symbols). There was no significant relationship for EXI (r = 0-06; P > 0-05) or for EX2 (r = 0-36; P > 0-05). exercise bouts, the lactate production could account for 67-2 (EXI-C) and 56-3% (EXI-HG) of the total anaerobic energyv release, while the corresponding values for EX2-C and EX2-HG were 45-7 and 36-4%, respectively (Fig. 6).

MUSCLE GLYCOGEN AND ANAEROBIC ENERGY YIELD

219

DISCUSSION

The present study gives a clear answer to the question of whether initial muscle glycogen content and the glycolytic rate are coupled in intense short-lasting exercise. Glycolysis, neither as a peak rate, nor as total contribution of energy during the exercise, is a function of above-normal muscle carbohydrate storage. Further, this relates both to its 'aerobic' and 'anaerobic' usage. The bulk of data in the literature on man are in line with our findings. Conflicting results, however, are also available and the question is then if these differences in results can be explained. Asmussen et al. (1974), and later Klausen & Sj0gaard (1980) and Maughan and colleagues (Maughan & Poole, 1981; Greenhaff et al. 1987, 1988), have all reported effects of dietary manipulation on blood lactate levels after short-lasting intense exercise. In the study by Klausen & Sj0gaard (1980) muscle analyses were also performed. In this study the diets produced differences in carbohydrate storage of the muscle (from approximately 60 to 140 mmol (kg wet wt)-1). Muscle and blood lactate levels, after repeated 2 min, exhaustive exercise bouts, were reduced in relation to the lowering of the muscle glycogen content. Of very special note is their finding of muscle glycogen depletion being identical (12-5 mmol (kg wet wt)-1) for each 2 min exercise bout (this is clearly pointed out by the authors in their discussion). This implies very similar rates of glycolysis. A possible explanation for the lower lactate concentration does not appear to be a reduced production, but rather an elevated utilization of the produced lactate in various tissues of the body when the carbohydrate storage was scarce. Muscle glycogen levels were surprisingly high after the fat and protein diet in this particular study. It is likely, however, that glycogen in the various fibres was unevenly distributed, which became further pronounced after the first exercise bout, and resulted in an elevated lactate turnover. In addition, it should be considered that several days with an extreme diet may enhance metabolic pathways for the use of lactate in oxidative metabolism (Jansson, 1980). Several investigations are consistent with the present experiment. Jacobs and colleagues (Jacobs, 1981; Symons & Jacobs, 1989), in a series of experiments comparing a large range of muscle glycogen contents, have been unable to demonstrate an effect on the rate of glycogen depletion during short very intense exercise. Ren et al. (1990) made similar findings in a study using electrical stimulation of the human quadriceps muscles. In contrast, there is another study in the literature where elevated muscle glycogen content was associated with an enhanced rate of glycogenolysis and improved performance. The larger carbohydrate storage was brought about by a period of sprint training and an ordinary mixed diet. The explanation for the observed alterations is likely to be related to the conditioning rather than to the small elevation in muscle glycogen stores; a conclusion also drawn by the authors (Boobis, Williams & Wootton, 1983; Boobis, 1986). Our conclusion, therefore, is that above-normal muscle glycogen content, and glycogenolytic and glycolytic rates in intense exercise, are not coupled. Differences in muscle and blood lactate concentrations which are sometimes found, can be attributed to the lactate being metabolized at different rates. Grisdale, Jacobs & Cafarelli (1990) have reported that exercise 24 h prior to repeated static contractions is a more potent determinant of muscle endurance than glycogen availability.

220

J. BANGSBO, T. E GRAHAM, B. KIENS AND B. SALTIN

However, when our subjects were tested on a separate day without any long-term exhaustive exercise the day before, performance time and glycogenolytic and glycolytic rates were the same (Fig. 8). Thus, in the present experiments muscle glycogen content can be singled out as the sole factor studied. 7

10 X.7

6

WE

°'

12 EoT LE 4--

8

CL

00

Ef

6

O'60

80 100 120 140 160 180 Pre-exercise glycogen. (mmol (kg wet wt-1) Fig. 8. Net lactate production rate (lower panel) and muscle glycogen utilization rate (upper panel) during EXI related to pre-exercise muscle glycogen concentration for four subjects, who performed the first exhaustive exercise on a separate day.

It is more difficult to explain the findings from rat models, which in part differ from man. Richter & Galbo (1986) found, when using an isolated rat hindlimb model and electrically inducing contractions for 15 min, that a coupling existed between content of glycogen in muscle and the rate by which it was utilized, particularly in fast twitch oxidative (FO) and fast twitch oxidative and glycolytic (FOG) fibres. Spriet et al. (1990) raised the question as to whether this was due to the welldocumented coupling of aerobic consumption to carbohydrate in more prolonged exercise. In experiments where circulation was occluded, they found a pronounced drop in tension development with intermittent stimulation of the muscle for 60 s. However, glycogen depletion and rate of lactate production were similar regardless of initial muscle glycogen content. Thus, they concluded that the findings by Richter & Galbo (1986) were compatible with their original suggestion (Spriet et at. 1990). Hespel and Richter re-studied this problem using their experimental model, and included muscle samples after 1 and 2 min of stimulation (personal communication). Both early and late in the stimulation period of 15 min, glycolysis was observed to be a function of the magnitude of the muscle glycogen storage. They attributed the linking of high muscle glycogen and high rates of glycolysis to a high content of phosphorylase a. Its activity at rest was positively related to the muscle glycogen content.

MUSCLE GLYCOGEN AND ANAEROBIC ENERGY YIELD 221 Electrically induced contractions mimic voluntary muscle activity; however, there are certain marked differences. One is the recruitment pattern, which with electrical stimulation favours involvement of the fast twitch (FT) fibres (Baratta, Ichie, Hurring & Solommon, 1989). This is probably not a serious concern in shortlasting experiments, where fatigue develops within 1-2 min, as the FT fibre pool in voluntary contractions also contributes the most in these circumstances. In exercise lasting 15 min, as in the case of Richter and colleagues, a 'reversed' recruitment order is obtained which means that comparisons with voluntary contractions are not valid. In the former situation, the FT pool of fibres is engaged early in the exercise for force production, but drops out quickly. For the remaining time, force production relates to slow twitch (ST) fibre tension development. Thus, the two major types of muscle fibres are studied separately, with a major impact of FT muscle fibre metabolism during the 1 and 2 min samples, whereas ST muscle response is manifested later in the exercise. This, by itself, is not an explanation for the linkage between glycogen content and its degradation. However, as a result of the supramaximal stimulation that was applied, it is likely that a high concentration of inorganic phosphate (Pi) develops, especially in fast twitch muscles. Chasiotis (1983, 1988) and later Ren & Hultman (1990) have shown that Pi is a critical activator of phosphorylase b to a, and subsequently of the rate of glycogen utilization. A likely explanation for the difference in results, comparing Richter and colleagues' findings with those of Spriet et al. as well as those on man, could be the difference in the activation of muscle. Supramaximal activation may cause markedly higher free Pi levels in the contracting muscle. It has been demonstrated in vitro that the enzyme glycogen phosphorylase has a very low Michaelis constant (Km 2 mm) for glycogen (Newsholme & Leech, 1983). In the present study the lack of relationship between initial muscle glycogen and glycogenolytic rate suggests that phosphorylase is saturated with its substrate at least when muscle glycogen is higher than 30-40 mmol (kg wet wt)-f. In several studies higher muscle IMP has been found when glycogen is low, which we also observed (Norman, Sollevi & Jansson, 1988; Broberg & Sahlin, 1989; Spencer & Katz, 1991). In addition, for the control leg muscle IMP concentration was correlated to muscle glycogen concentration at exhaustion both for EXI and EX2 (r = 0 77 and r = 0X83, respectively, P < 0-05), and to the relative number of almost glycogendepleted muscle fibres after EXI-C determined by PAS staining (r = 0-78, P < 0 05). The elevated IMP, which may signify more of the ADP and AMP being unbound, would aid in maintaining the glycogen breakdown and formation of pyruvate when muscle glycogen and glycogen-6-phosphate are low in the muscle (Uyeda, 1979; Aragon, Tornheim & Lowenstein, 1980; Chasiotis, Sahlin & Hultman, 1982). A reduction in muscle glycogen content was also achieved by prior exercise (i.e. EXI), followed by a long enough recovery period (1 h) for acid-base balance to become normalized. Only 26 mmol (kg wet wt)-1 was utilized by the first exercise, and during the 1 h recovery period about 12 mmol (kg wet wt)-1 was synthesized in part using lactate as a substrate (Bangsbo, Gollnick, Graham & Saltin, 1991). Thus, the difference in muscle glycogen prior to EXt and EX2 was small, amounting to about 14 mmol (kg wet wt)-1, with no subject having less than 34 mmol (kg wet wt)-1 prior to EX2 and all fibres containing glycogen (Fig. 3). In the light of the present %

222

J. BANGSBO, T. K GRAHAM, B. KIENS AND B. SALTIN

results which indicate no role for muscle glycogen over the range of 30-250 mmol (kg wet wt)-' for anaerobic metabolism and performance, the results when exercise was performed a second time are striking. Both lactate production and performance were lowered when exercising with the control leg, but there was no decrease in performance for the high-glycogen leg (EX2-HG; Fig. 9). EX2 t14 0

EXi

-

126

4

)col

0

z EE

Control High Control High glycogen glycogen Fig. 9. Net muscle glycogen utilization rate during EXI (left) and EX2 (right) related to lactate production rate (0), and carbohydrate oxidation rate ([[I) for the control leg

(C) and the high-glycogen leg (HG) exercises.

(E1, others). It is assumed that leg RQ = 1 during the

What brought about the lowering of the rate of glycolysis in EX2? The IMP concentration was the same comparing EXI and EX2. Breakdown of ATP and elevation of ADP, AMP and probably also Pi were very similar in all four exercise bouts, but it is difficult to propose a mechanism other than a change in bound to unbound ratio for these regulators acting at the level of phosphofructokinase (PFK) during the second exercise (EX2-C and EX2-HG), with this ratio changing towards less being unbound. The reason for suggesting that regulation is at the level of PFK rather than at the conversion of phosphorylase a to b, is that glycogenolysis was not lowered in EX2-HG and that glucose-6-phosphate accumulation appears to be a function of muscle glycogen content (Hultman & Sj0holm, 1983; Spencer & Katz, 1991). The explanation for less lactate being accumulated and released from the active muscles cannot be an elevated turn-over within the muscle. Instead, the most likely explanation is that the formation of lactate is the net result of the rate of pyruvate and NADH production in the cytosol, and their uptakes by the mitochondria, the latter appearing to be unaltered in the present experiments. Lactate dehydrogenase is a near-equilibrium enzyme and so its flux is dictated by the concentrations of substrates and products. The smaller production of three-carbon skeleton via glycolysis would then result in less lactate being produced. Muscle and blood lactate (and blood pH) at exhaustion were lower in EX2 than in EX1, which supports the notion that lactate and pH are not the only crucial 'fatigue

MUSCLE GLYCOGEN AND ANAEROBIC ENERGY YIELD 223 factors' (Edwards, 1983). In addition, nucleotide and CP metabolism were not affected. It should be emphasized that with the exercise model used in the present experiment, the site for fatigue is local. The systemic effects of one-legged dynamic knee extensor exercise are minor. Whole-body oxygen uptake is less than one-third of the subject's maximal oxygen uptake, heart activity is 120-140 beats/min and catecholamines are low (Bangsbo et al. 1990). Although rate of lactate production is high in the active muscle, and its release to the blood stream is high, elevations in systemic circulation are only 5-6 mm. Blood glucose is maintained above 4-5 mm. At point of exhaustion, defined as the inability to maintain the pre-set kicking rate (1 Hz), clear indications of fatigue were already present. Force recordings revealed a reduction in amplitude which was compensated for by an elongated contraction. Further, a tendency towards pushing the leg back into its vertical position was sometimes apparent the closer the exercise came to the point of exhaustion. Thus, definite objective signs of muscle fatigue were present. In this connection, it should be noted that there was no 'transfer' effect of fatigue, i.e. when the exercise was performed with leg 2 after leg 1 had exercised twice, time to exhaustion was identical when comparing EXI with the control and HG leg. These data speak in favour of prior exercise causing an excitation-contraction failure to develop faster when intense exercise is repeated. It is difficult to believe that the mechanism is related to a K+/Na+ imbalance across the sarcolemma or in the T-tubuli system as the normalization of these ions, although slow after a very fast early recovery, would have been completed by an hour of rest (Juel, 1986; Juel, Bangsbo, Graham & Saltin, 1990). Although somewhat less K+ was released during EX2-HG, femoral venous K+ concentrations were very similar in all exercise bouts. If this K+ concentration reflects the interstitial concentration, it seems that exhaustion occurs at a well-defined K+ imbalance in the muscle. Fatigue at the level of cross-bridge interaction has also been reported (Edman, 1991), but there are no data indicating that it persists for very long. Two more possible causes should be considered, and they have in common that complete recovery after exhaustive exercise takes time. The Ca2+ kinetics of the SR system are affected by intense, exhaustive exercise with a marked slowing of the Ca2+ uptake, related to elongation of the half-relaxation time, and also a drop in maximal force development (Gollnick, Korge, Karpakka & Saltin, 1991). The time course for its normalization is more than 30 min, but exactly how long is unknown. The other possibility would be that a reduction in neural drive develops faster in EX2. It is unlikely that it is related to 'low frequency fatigue', as EXI was very intense and short lasting. The same relates to recent findings that after prolonged exhaustive exercise the EMG activity was reduced in relation to the drop in maximal force development, which persisted long into recovery (Nicol, Komi & Marconnet, 1991). A more likely explanation may be that of a reduced nervous drive due to reflex inhibition at the spinal level as demonstrated by Garland & McComas (1990). Glycogen content may have a role, as performance was unaltered when exercising a second time with the high-glycogen leg (EX2-HG). Possible mechanisms linking the two phenomena are presently unknown. In agreement with our previous observation (Bangsbo et al. 1990), the energy release from lactate production and utilization of CP and nucleotides during the first

224

J. BANGSBO, T. E. GRAHAM, B. KIENS AND B. SALTIN

exercise could account for 80-90 % of the total anaerobic energy production (Fig. 6). During the second exercise bout (EX2-C and EX2-HG) the alteration in these variables could only account for 60-70 % of the total anaerobic energy release. The lower glycolytic rate during EX2 indicates that the accumulation of three-carbon glycolytic intermediates is less during EX2. Similarly, it seems unlikely that the difference is related to a difference in the underestimation of the lactate production during EX2 due to lactate uptake by the knee flexor muscles, as the arterial blood lactate concentration was the same during EXI and EX2. Another possibility could be that the energy demand during EX2 was lower compared to EX1 due to either a longer contraction time, although the power was the same (Kushmerick, 1985; di Prampero, Boutellier & Marguerat, 1988), or due to changes in factors influencing the free energy (AG) for ATP hydrolysis, such as increase in temperature, Pi and reduction in pH (Kawai, Guth, Winnikes, Haist & Ruegg, 1987; Cooke, Franks, Luciani & Pate, 1988). There was no indication of difference in the contraction pattern between EXt and EX2, no difference in muscle temperature and probably none in free Pi. However, the lower muscle lactate at the end of EX2 indicated a higher muscle pH, which might have resulted in a higher energy release from ATP breakdown during EX2. If this was the case the estimated oxygen deficit could overestimate the true ATP production. Whether this factor can quantitatively account for the difference between EXt and EX2 is, however, doubtful. Additional studies of the mechanical efficiency of in vivo muscle contractions, including possible variations in AG for the ATP hydrolysis, are needed to clarify this problem. In conclusion we have found no coupling between above-normal muscle glycogen storage, enhanced glycogenolysis, glycolysis and performance. Prior, short, exhaustive exercise with only a small glycogen utilization followed by time for normalization of the acid-base balance of the muscle, caused a significant reduction in performance when the exercise was performed the second time. Glycogenolysis was also reduced and so was lactate production, but not the aerobic utilization of pyruvate and its contribution to ATP resynthesis. The study was supported by grants from Team Danmark and Danish Natural Science Foundation (11-7776). Terry Graham was supported by an NSERC (Canada) international collaborative research grant. REFERENCES

ANDERSEN, P. (1975). Capillary density in skeletal muscle of man. Acta Phy8iologica Scandinavica 95, 203-205. ANDERSEN, P., ADAMS, R. P., SJ0GAARD, G., THORBOE, A. & SALTIN, B. (1985). Dynamic knee extension as a model for the study of an isolated exercising muscle in man. Journal of Applied Phyaiology 59, 1647-1653. ANDERSEN, P. & SALTIN, B. (1985). Maximal perfusion of skeletal muscle in man. Journal of Phy8iology 366, 233-249. ARAGON, J. J., TORNHEIM, K. & L6WENSTEIN, J. M. (1980). On a possible role of IMP in the regulation of phosphorylase activity in skeletal muscle. FEBS Letter8 117, K56-64. ASMUSSEN, E., KLAUSEN, K., NIELSEN, L, EGELUND, L., TECHOW, 0. S. A. & ToNDER, P. J. (1974). Lactate production and anaerobic work capacity after prolonged exercise. Acta Physiologica Scandinavica 90, 731-742.

MUSCLE GLYCOGEN AND ANAEROBIC ENERGY YIELD

225

BANGSBO, J., GOLLNICK, P. D., GRAHAM, T. E., JUEL, C., KIENS, B., MIzuNo, M. & SALTIN, B. (1990). Anaerobic energy production and 02 deficit - debt relationship during exhaustive exercise in humans. Journal of Physiology 422, 539-559. BANGSBO, J., GOLLNICK, P. D., GRAHAM, T. E. & SALTIN, B. (1991). Substrates for muscle glycogen synthesis in recovery from intense exercise in man. Journal of Physiology 434, 423-440. BARATTA, R. U., ICHIE, M., HURRING, S. & SOLOMMON, N. I. (1989). A method for studying muscle properties under orderly stimulated motor units with tripolar nerve cuff electrodes. Journal of Biomedical Engineering 11, 141-147. BERGSTROM, J., HERMANSEN, L., HULTMAN, E. & SALTIN, B. (1967). Diet, muscle glycogen and physical performance. Acta Physiologica Scandinavica 71, 140-150. BOOBIS, L. H. (1987). Metabolic aspects of fatigue during sprinting. In 'Exercise. Benefits, Limits and Adaptations, ed. MACLEOD, D., MAIJGHAN, R., NIMMO, M., REILLY, T. & WILLIAMS, C., pp. 116-143. E & F. N. Spon, London/New York. BOOBIS, L. H., WILLIAMS, C. & WOOTTON, S. A. (1983). Influence of sprint training on muscle metabolism during brief maximal exercise in man. Journal of Physiology 342, 36-37P. BROBERG, S. & SAHLIN, K, (1989). Adenine nucleotide degradation in human skeletal muscle during prolonged exercise. Journal of Applied Physiology 67, 116-122. BROOKE, M. H. & KAISER, K. K. (1970). Three "myosin adenosine trisphosphatase" systems: The nature of their pH lability and sulphydryl dependence. Journal of Histochemistry and Cytochemistry 18, 670-672. BROUN, B. W. & HOLLANDER, M. (1977). Statistics. A Biomedical Introduction. Wiley, New York. CHASIOTIS, D. (1983). The regulation of glycogen phosphorylase and glycogen breakdown in human skeletal muscle. Acta Physiologica Scandinavica Supplementum 518, 1-68. CHASIOTIS, D. (1988). Role of cyclic AMP and inorganic phosphate in the regulation of muscle glycogenolysis during exercise. Medicine and Science in Sport and Exercise 20 (6), 545-550. CHASIOTIS, D., SAHLIN, K. & HULTMAN, E. (1982). Regulation of glycogenolysis in human muscle at rest and during exercise. Journal of Applied Physiology 53 (3), 708-715. CHRISTENSEN, E. H. & HANSEN, 0. (1939). Arbeitsfahigkeit und ernahrung. Skandinavische Archiv

fur Physiologie 81, 160-171. COOKE, R., FRANKS, K., LuCIANI, G. B. & PATE, E. (1988). The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate. Journal of Physiology 395, 77-97. DI PRAMPERO, P. E., BOUTELLIER, U. & MARGUERAT, A. (1988). Efficiency of work performance and contraction velocity in isotonic tetani of frog sartorius. Pfiuigers Archiv 412, 455-461. DRABKIN, D. L. & AUSTIN, J. H. (1935). Spectrophotometric studies II. Preparations from washed blood cells, nitric oxide hemoglobin and sulfhemoglobin. Journal of Biological Chemistry 122, 51-65.

EDMAN, K. A. P. (1991). The contractile performance of normal and fatigued skeletal muscle. In Fatigue Mechanisms in Exercise and Training, ed. MARCONNET, P., KoMI, P., SALTIN, B. & SEJERSTED, 0. S. Karger, Basel, Miinchen, Paris, London and Sydney. EDWARDS, R. H. T. (1983). Biochemical bases of fatigue in exercise performance: Catastrophe theory of muscular fatigue. In Biochemistry of Exercise, ed. KNUTTGEN, H. C., VOGEL, J. A. & POORTMANS, J., pp. 3-29. Human Kinetics, Champeign, IL, USA. GARLAND, S. J. & MCCOMAS, A. J. (1990). Reflex inhibition of human soleus muscle during fatigue. Journal of Physiology 429, 17-27. GOLLNICK, P. D., K6RGE, P., KARPAKKA, J. & SALTIN, B. (1991). Elongation of skeletal muscle relaxation during exercise is linked to reduced calcium uptake by the sarcoplasmic reticulum in man. Acta Physiologica Scandinavica 142, 135-136. GREENHAFF, P. L., GLEESON, M. & MAUGHAN, R. J. (1987). The effects of dietary manipulation on blood acid-base status and the performance of high intensity exercise. European Journal of Applied Physiology 56, 331-337. GREENHAFF, P. L., GLEESON, M. & MAUGHAN, R. J. (1988). The effects of a glycogen loading regimen on acid-base status and blood lactate concentration before and after a fixed period of high intensity exercise in man. European Journal of Applied Physiology 57, 254-259. GRISDALE, R. K., JACOBS, I. & CAFARELLI, E. (1990). Relative effects of glycogen depletion and previous exercise on muscle force and endurance capacity. Journal of Applied Physiology 69 (4), 1276-1282.

226

J. BANGSBO, T. E. GRAHAM, B. KIENS AND B. SALTIN

HOLMGREN, A. & PERNOW, B. (1959). Spectrophotometric determination of oxygen saturation of blood in the determination of cardiac output. Scandinavian Journal of Clinical Laboratory Investigation 11, 143-149. HULTMAN, E. & SJ0HOLM, H. (1983). Substrate availability. In Biochemistry of Exercise, ed. KNUTTGEN, H. C., VOGEL, J. A. & POORTMANS, J. pp. 63-75. Human Kinetics, Champeign, IL, USA. JACOBS, I. (1981). Lactate concentrations after short, maximal exercise at various glycogen levels. Acta Physiologica Scandinavica 111, 465-469. JANSSON, E. (1980). Muscle enzyme adaptation to diet in man. In Biochemistry of Exercise 13, 299. JONES, P. R. M. & PEARSON, J. (1969). Anthropometric determination of leg fat and muscle plus bone volumes in young male and female adults. Journal of Physiology 204, 36P. JUEL, C. (1986). Potassium and sodium shifts during in vitro isometric muscle contractions, and the time course of the ion-gradient recovery. Pflugers Archiv 406, 458-463. JUEL, C., BANGSBO, J., GRAHAM, T. & SALTIN, B. (1990). Lactate and potassium fluxes from skeletal muscle during intense dynamic knee-extensor exercise in man. Acta Physiologica Scandinavica 140, 147-159. KAWAI, M., GUTH, K., WINNIKES, K., HAIST, C. & RUEGG, J. C. (1987). The effect of inorganic phosphate on ATP hydrolysis rate and the tension transients in chemically skinned rabbit psoas fibers. Pflugers Archiv 408, 1-9. KLAUSEN, K. & SJ0GAARD, G. (1980). Glycogen stores and lactate accumulation in skeletal muscle of man during intense bicycle exercise. Scandinavian Journal of Sports Science 2, 7-12. KUSHMERICK, M. J. (1985). Patterns in mammalian muscle energetics. Journal of Experimental Biology 115, 165-177. LowRY, 0. H. & PASSONNEAU, J. V. (1972). A Flexible System on Enzymatic Analysis. Academic Press, New York. MAUGHAN, R. J. & POOLE, D. C. (1981). The effects of a glycogen loading regimen on the capacity to perform anaerobic exercise. European Journal of Applied Physiology 46, 211-219. NEWSHOLME, E. A. & LEECH, A. R. (1983). Biochemistry for the Medical Sciences. John Wiley and Sons, New York. NICOL, C., KoMI, P. V. & MARCONNET, P. (1991). Fatigue effects of marathon running on neuromuscular performance. II. Changes in force, integrated electromyographic activity and endurance capacity. Scandinavian Journal of Medicine and Science in Sports 1 (1), 18-24. NORMAN, B., SOLLEVI, A. & JANSSON, E. (1988). Increased IMP content in glycogen-depleted muscle fibres during submaximal exercise in man. Acta Physiologica Scandinavica 133, 97-100. PADYKULA, H. A. & HERMAN, E. (1955). The specificity of the histochemical method for adenosine triphosphate. Journal of Histochemistry and Cytochemistry 3, 170-183. REN, J. M., BROBERG, G., SAHLIN, K. & HULTMAN, E. (1990). Influence of reduced glycogen level on glycogenolysis during short-term stimulation in man. Acta Physiologica Scandinavica 139, 467-474. REN, J. M. & HULTMAN, E. (1990). Regulation of phosphorylase a activity in human skeletal muscle. Journal of Applied Physiology 69 (3), 919-923. RICHTER, E. A. & GALBO, H. (1986). High glycogen levels enhance glycogen breakdown in isolated contracting skeletal muscle. Journal of Applied Physiology 61, 827-831. RICHTER, E. A., MIKINES, K. J., GALBO, H. & KIENS, B. (1989). Effect of exercise on insulin action in human skeletal muscle. Journal of Applied Physiology 66, 876-885. SALTIN, B. (1985). Hemodynamic adaptations to exercise. American Journal of Cardiology 55, 42-47D. SALTIN, B. & HERMANSEN, L. (1967). Glycogen stores and prolonged severe exercise. In Symposium of Swedish Nutrition Foundation, ed. BLIX, G., pp. 32-46. Almquist and Wiksell, Uppsala. SALTIN, B., KIENS, B. & SAVARD, G. (1986). A quantitative approach to the evaluation of skeletal muscle substrate utilization in prolonged exercise. In Biochemical Aspects of Physical Exercise, ed. BENZI, G., PACKER, L. & SILIPRANDI, N., pp. 235-244. Elsevier Science Publisher B.V. SCHWEINSBERG, P. D. & Loo, T. L. (1980). Simultaneous analysis of ATP, ADP, AMP and other purines in human erythrocytes by HPLC. Journal of Chromotography 181, 103-107. SHIMIZU, S., INOUE, K., TANI, Y. & YAMADA, H. (1979). Enzymatic microdetermination of serum free fatty acids. Analytical Biochemistry 98, 341-345. SIEGEL, S. (1965). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York.

MUSCLE GLYCOGEN AND ANAEROBIC ENERGY YIELD

227

SPENCER, M. K. & KATZ, A. (1991). Role of glycogen in control of glycolysis and IMP formation in human muscle during exercise. American Journal of Phy8iology 260, E859-864. SPRIET, L. L., BERARDINUCCI, L., MARSH, D. R., CAMPBELL, C. B. & GRAHAM, T. (1990). Glycogen content has no effect on skeletal muscle glycogenolysis during short-term tetanic stimulation. Journal of Applied Phy8iology 68 (5), 1883-1888. SYMONS, J. D. & JACOBS, I. (1989). High-intensity exercise performance is not impaired by low intramuscular glycogen. Medicine and Science in Sport8 and Exerci8e 21 (5), 550-557. UYEDA, K. (1979). Phosphofructokinase. Advance8 in Enzymology and Related Area8 of Molecular Biology 48, 193-244.